It has been sufficiently evident that poly- or oligonucleotides of sensible (sense and/or antisense) sequences can be used as effective therapeutic agents in drug therapy, vaccination and tissue regeneration by turning the relevant gene on (expression) or off (silencing)[1]. To achieve such therapeutic efficacy, however, therapeutic genes, DNA vaccines as well as siRNA drugs must be delivered to the nuclei or cytoplasm of target cells. Among the carrier systems for delivering polynucleotides (also named as gene, gene materials, oligonucleotides, nucleic acids hereafter or DNA and RNA), synthetic delivery systems possess a series of advantages over viral vectors such as freedom from immunity and viral mutation, ability to package multiple genes or siRNA of choice into particulate vehicles via a single mechanism, and adaptability to simple and cost-efficient manufacturing process[2]. To deliver gene materials (DNA and RNA) to targeted inter- and intra-cellular sites effectively using non-viral systems, the synthetic gene carriers (i.e. non-viral vectors) must accomplish a number of tasks consequently comprising (A) packing gene materials to nano-particulate forms to avoid pre-phagocytosis degradation, (B) attaching on targeted cells selectively, (C) facilitating endosomal escape of gene materials, (D) releasing gene materials in cytoplasm, and (E) metabolizing itself to nontoxic species. There is yet, however, a synthetic gene delivery system reported to date meets all these criteria.
Synthetic gene delivery vehicles reported in last decades can, in general, be divided into several categories, cationic liposome-based systems (called lipoplex), cationic polymer-based systems (called polyplex), lipid-cationic polymer combined systems (called lipopolyplex) and non-charged nanometer particulates. The majority of them are lipoplexes and polyplexes due to the negative charges of DNA and RNA by which the gene materials may easily be condensed into particles with positively charged liposomes or polymers. These two categories possess different advantages and mechanisms in terms of each step of gene transfection. Cationic liposomes condense gene materials less compactly than cationic polymers[3] but offer unique membrane fusion function with endosomes that may help DNA or RNA to escape to cytoplasm in molecular form[4]. Polycations (cationic polymer), on the other hand, may condense gene materials in more compacted forms[3] so that better protection and larger capacity of gene materials are expected[5]. For endosomal escaping, polyplex is believed to undergo a “proton sponging” process for which the polyplex-engulfing endosome is ruptured by chloride ions accumulated due to continuous influx pumping of HCl into endosomes to compensate the protons consumed by the cationic polymer carrier. In this case, the protonated polycation gains more positive charges to bind DNA or RNA due to which the gene materials enter cytoplasm in the form of particles rather than molecules. It has been reported that ruptured endosomes may be self-repaired so that the polyplex particle may be re-encapsulated before escaping[6]. In addition, the polynucleotides must be released or extracted out of the polyplex in order to exert their biological functions. It seems that condensation and release of DNA or RNA by polycations are a pair of contradictory processes which require a polycationic carrier system to be chemically dynamic and biologically responsive.
To compromise gene packing and release, some researchers suggested to use or design a polycationic carrier which possesses a mild strength of gene condensation[7]. Using a cationic polymer with low molecular weight or with low amino group density is one of the approaches[8]. Another strategy is to use environment responsive polycations to achieve gene condensation and releasing, the two opposite moves, consequently[9]. This type of polymers are, however, often complex in structures and complicated in metabolic process and metabolized products. Using degradable cationic polymers as gene carriers may be a more reasonable approach by which gene release may be achieved by degradation of the backbone of the carriers, a process independent of its ability to condense DNA or RNA[10]. Degradation to small molecules will reduce chemical toxicity of polycations. As reported in the literature, biodegradable linkages such as carboxylic ester, phosphate ester, imine or disulfide structure were incorporated in the backbone of a cationic polymers. In this aspect, ester bond is the most widely used degradable structure to incorporate into the polycation backbone for its balanced stability and degradability. However, ester bond is highly reactive to nucleophiles such as primary and secondary amino groups[11], which are the key functional groups for gene compacting and proton sponging. In addition, degradation of ester structures creates acids that compromise the proton-sponging effect.
Some researchers polymerized branched small molecular polyethylenimine (PEI) via an ester-bearing linker, and the cross-linked small molecular PEI carriers possess higher gene transfection efficiency but lower toxicity[14]. Backbone degradation of this polymer was achieved by cleavage of the linker, leaving the cleft fragments attached to the small molecular PEI or other amino group-bearing monomers (the polymer building blocks)[12-14]. Such a backbone degradation pattern may be fine for a polycationic gene carrier formed of man-made amino group-bearing building blocks. For a degradable cationic polymer formed of endogenous amino group-bearing monomers, the attachment of linker fragments upon polymer degradation will dismiss the advantages of using endogenous monomers. A polycationic gene carrier degradable to human endogenous amino group-bearing monomers is an ideal design to achieve intracellular release of genes and metabolic elimination of the carrier itself.
WO2009/100,645 disclosed a method to develop polycationic gene carriers which possess sufficient amount of amino groups to condense polynucleotides into compacted particles and to induce endosomal break through proton sponge effect[15], and possess fully degradable backbone to release polynucleotides after endosomal escape and to turn itself to endogenous or non-toxic metabolites.